This invention relates, in general, to slide loading devices for use in automated cell counting systems or standard optical microscopes. More particularly, the invention relates to a slide holder that exerts a uniform force on a coverslip ensuring a uniform volume of a sample on the slide.
Accurate measurement of cell concentration in a biological sample is important in a variety of medical fields. For example, the concentration of leukocytes in blood or sperm cells in semen serve as diagnostic indicators. In particular the concentration of sperm cells is a direct indicator of fertility. In this situation the concentration may vary from zero to more than 100 million cells per milliliter. In response to the need for accurate measurement, a variety of devices have been proposed or developed for measuring cell concentration.
One type of apparatus for measuring the concentration of cells is a flow cytometer in which cells are individually counted as they emerge in a stream from an orifice. Knowing the flow rate allows the technician to calculate the cell concentration. Due to the size of the orifice which is necessary to govern low, the orifice is subject to blockage and other problems, requiring highly skilled operation and care of this expensive and delicate device.
An alternative method has been used to measure the concentration which involves measuring the area density of cells in a preparation of known depth. A chamber is defined by grid lines drawn in squares and has a known depth (usually 10-1000 .mu.m). Knowing the chamber depth and counting the number of cells per unit area in the grid gives the concentration. The accuracy of this measurement is proportional to the accuracy with which the chamber depth is known.
Various apparatus configurations have been devised to accommodate this method. The Hemacytometer uses a glass coverslip suspended over a specimen plate, the gap being filled by capillary action. This system uses a chamber depth of approximately 100 .mu.m which is too large for highly concentrated fluids such as semen. Additionally, to count such fluids with motile cells, a lethal diluent must be used to immobilize the cells. This, naturally, prevents the observation of the moving cells. Because the 100 .mu.m chamber depth is greater than the focal depth of the typical objective lens system employed, a smaller chamber depth is required.
Another device that uses the above method is the Makler chamber. This device has a weighted coverslip suspended on four posts approximately 10 .mu.m above the specimen plate. The weight of the coverslip forces the specimen to spread out to a depth of 10 .mu.m. Using this device can result in considerable over-estimations of concentration because the viscosity of the fluid specimen impacts the fluid's ability to spread. Also, if a solid particle should become lodged under the coverslip, the depth is affected, again skewing the measurement.
A third device that uses the above method is the Microcell. It uses two fixed planes of glass to achieve a separation distance of 20 .mu.m and is filled by capillary action. Since the distance between the planes can be measured, the chamber depth is known. In this device, viscous samples are unable to fill the chamber by capillary action. Also, since precise measurement of the separation distance between the two glass planes is required to know the chamber depth, the preparation of the device is expensive. Capillary action further risks damaging fragile cells such as sperm due to the fluid flow causing a tumbling motion during loading.
A simpler alternative was developed which uses a printed ink pattern on a slide to define an area of constant depth. Placing a coverslip over it provides a chamber of known depth permitting concentration to be measured. This alternative overcomes most of the aforementioned problems, but introduces another. Unless the coverslip is maintained at the correct distance from the slide, the chamber distance will not be constant between samples. Too much sample will cause the coverslip to rise, while too little will distort the slip inwardly through capillary action. Any such distortion, at a 20 .mu.m depth, is significant enough to give erroneous results.
Accordingly, there exists a need for a slide loading device which can provide accurate and reliable measurement and observation of a biological sample, regardless of the viscosity of the fluid.
It is another object of the invention to provide a slide loading device which yields accurate measurement of cell concentration, independently of the amount of sample in the chamber.
Other general and specific objects of the invention will in part be obvious and will in part appear hereinafter.